DRAFT: This module has unpublished changes.
Lab report 1. Fundamental Lab Skills - Serial Dilution, Dilutions and Solution Prep
Name: Igor Popovich
Lab partner: Lada Grigorieva
Professor: Paul Kasili, Ph.D.
Class: BIO 208-01
Date: 1 February 2016
Abstract: The basic techniques in a cell and molecular biology are aseptic technique, dilution, and colony streaking, which are crucial for laboratory works. Our major purpose of this experiment is to learn these techniques and observe them in a practice. We obtained Staphylococcus aureus broth culture as a sample for our “preparation of plates from tubes 4D, 5D, and 6D” and “preparing a streak plate” experiments. The first part of the laboratory work showed that the more we dilute, the less number of bacteria colonies i.e. CFU/ml we can observe after spreading and incubating them on a plate. The obtained results showed a little deviation for a fifth diluted tube due to less pipetted volume from the tube, but the overall results confirmed that the dilution factor was preserved till the last dilution. So, we could calculate the concentration of bacteria in the original culture. After the second part of the experiment, we isolated bacteria colonies by colony streaking technique and could study them further if we needed. The description of obtained bacterial colonies was similar to the description of Staphylococcus aureus. Thus, we have learned how to properly use these three techniques, observed the difference of grown bacteria on plates after several dilutions, and got isolated colonies of bacteria.
Introduction: Aseptic technique, dilution, and colony streaking are frequent experiments of microbiologists. These techniques help us to clean or get rid of contaminations. Aseptic techniques are defined as techniques, which contribute to keep things free from contaminations, as for example, bacteria. This technique is always used when laboratory workers perform labs with living cell cultures. Also, aseptic technique requires usage of flame, which usually comes from Bunsen burner, to kill contaminating organisms. Colony streaking technique helps scientists to isolate individual colonies. Thus, scientists can separate pure strain of bacteria (for example) from other microorganisms in solution, so they can grow these bacteria on a new plate to study them (“Aseptic Technique, Dilution, Streaking, and Spread Plates,” 2015). Microbiologists use dilution techniques when they are required to perform experiment with using of small concentrations, making highly-diluted solutions. Usually the dilution factor after each dilution is constant, but results a geometric progression. For example, if we take 0.1 ml from original culture sample and transfer it into 0.9 ml tube with broth, we will get 1:10 dilution. If then, we transfer 0.1 ml from the diluted solution into a new 0.9 ml tube with broth, we will get 1:100 dilution because we had a 1:10 dilution, which we diluted again by dilution factor 1:10. By this serial dilution technique we can maximally reduce the colonies of bacteria. Also, using this technique, we can calculate the initial concentration of cells in the original culture, having counted the number of grown colonies of bacteria after the last dilution and dividing it by dilution factor (“Dilution Theory and Techniques,” 2003).
Purpose:The purpose of this experiment is to learn such fundamental skills as aseptic technique, dilutions, and colony streaking. Also, to investigate how serial dilution affects colonies of bacteria and to observe the decrease of number of colonies after each set of streaks Students should learn these techniques, as they are common and basic techniques in molecular and cell biology.
Hypothesis: My hypothesis for the first part of the experiment (preparation of plates from tubes 4D, 5D, and 6D) is that we will observe less number of bacteria colonies after each dilution, so the last plate from 6D sample with dilution factor of 1:1,000,000 will have the smallest number of bacteria colonies. For the second part of the experiment (preparing a streak plate) we will observe less colonies after each set of streaks, so we will have the smallest number of colonies (isolated colonies) on our fourth set of streaks.
Materials:
- Staphylococcus aureus broth culture
- 6 Nutrient broth tubes
- 4 Nutrient agar plates
- Sterile plastic loops
- Sterile plastic spreaders
- Sterile tips
- Micropipettors (P 20, P 200, P 1000)
- Bunsen burner
Procedure: Gloves were put on. The table was wiped down with a disinfectant. A Bunsen burner and a spark lighter were obtained; Bunsen burner was ignited. Six tubes with liquid broth were obtained and labeled as 1D, 2D, 3D, 4D, 5D, and 6D. Using the Staphylococcus aureus culture, serial dilutions were prepared according to the table 1 in a manual. As the dilutions were done, three nutrient agar plates (4D, 5D, 6D) were obtained and labeled. 100 μL from each of the tubes 4D, 5D, and 6D were aseptically transferred to three nutrient agar plates. Using sterile L-spreaders, these cultures were spread over the entire plate, so all the culture was absorbed in to the plate. After, the plates were incubated in the 37 Celsius degrees incubator. For the second part of the lab (preparing a streak plate), one agar plate was obtained and labeled. The Bunsen burner was ignited. The work was performed around the Bunsen burner. Using a sterile loop, the Staphylococcus aureus culture was removed from microfuge tube. Then, the culture was streaked on a nutrient agar plate. The loop was sterilized again, and the second set of streaks was performed. The loop was sterilized again, and the third set of streaks was performed. The last streak was done without sterilizing. The plate was incubated. On the next week, the plates were removed from the incubator, the colonies from 4D, 5D, and 6D were counted, and results were recorded.
Results:
Table 1: the number of colonies on 4D, 5D, and 6D agar plates.
Tube | Description of sample | Number of colonies obtained. CFU/100μL | Comments |
4D | Dilution number 4 | 376 | We divided the plate by a marker, so it facilitated our counting |
5D | Dilution number 5 | 23 | Not expected number of colonies (we expected around 40 colonies) |
6D | Dilution number 6 | 4 |
Figure 1 shows the obtained results after “preparation of plates from tubes 4D, 5D, and 6D” and “preparing a streak plate” experiments.
Discussion: To learn basic techniques in microbiology, we performed two experiments, “preparation of plates from tubes 4D, 5D, and 6D” and “preparing a streak plate” using Staphylococcus aureus culture. The overall results showed that the more we dilute the original culture, the less grown bacteria colonies we will observe on the plate. And the more sets of streaks we make, the less grown bacteria colonies we will observe.
Having performed the first part of the experiment, we obtained the results, which did not totally justify our expectations and which can be seen from the table 1. We observed that on the fifth plate with a dilution from tube number 5, we obtained 23 colonies, instead of expected 40. The reason might be that when we pipetted solution from the fifth diluted tube to spread the bacteria onto plate, air also could be pipetted with a desired solution. Having formed an air bubble, the volume decreased from the needed 100 μL and therefore, the amount of bacteria cells also decreased. Otherwise, we can observe the desired dilution of 1:100 between fourth and sixth tubes. As the fifth tube is an intermediate between those two tubes, we can say that the dilution procedure was correctly done. Thus, taking the average value of obtained results, we can calculate the concentration of bacteria in the original culture. As we can see, the sixth plate consists of four colonies per 100 μL and dilution factor is 1:1,000,000; so, the possible initial concentration of bacteria in the original is 4*10^7 CFU/ml. Following the same logic, we can calculate that the possible initial concentration of bacteria in the original culture, according to the fourth and fifth tubes, are 3.76*10^7 CFU/ml and 2.3*10^7 CFU/ml respectively. The average from these 3 tubes is 3.35*10^7 CFU/ml; so, we can assume that our original culture consists of 3.35*10^7 cells per ml. Knowing the dilution factor and the average concentration in the original culture, we can calculate the concentration of bacteria in tube 1D, tube 2D, and tube 3D. The dilution factor is 1:10, 1:100, and 1:1000 respectively, and we calculated that 3.35*10^6 CFU/ml in tube 1D, 3.35*10^5 CFU/ml in tube 2D, and 3.35*10^4 CFU/ml in tube 3D.
Having performed the second part of the experiment, we obtained the desired result and got isolated colonies (figure 1). The shape of these colonies is round, the margin is smooth (entire), the elevation is raised. This description is similar to the description of Staphylococcus aureus, which we were tried to isolate. Actually, having isolated these colonies from other organisms, we could perform a further research to study the bacteria.
Conclusion: Having performed the laboratory work, we learned aseptic technique, serial dilution, and colony streaking skills. The hypothesis was confirmed, having shown that each dilution decreases number of cells per ml, and that the more set of streaks we perform, the more isolated colonies we get. We observed how serial dilution affects colonies of bacteria (using the example of Staphylococcus aureus) having given rise to fewer colonies. So, we obtained 376 CFU/100 μL on 4D agar plate, 23 CFU/100 μL on 5D agar plate, and 4 CFU/100 μL on 6D agar plate. The reason of why we did not get the expected 40CFU/100 μL on 5D agar plate is that we pipetted less than required 100 μL to spread the volume on plate. However, the overall results confirmed that the dilution factor was preserved till the last dilution. The second part of the experiment showed that we isolated needed colonies of bacteria, as we could observe the decrease of number of colonies after each set of streaks. To improve “serial dilution experiment” I would use several plates (2-3) for each diluted tube, so we could obtain the average from these plates that could lead to more accurate results in calculations of the concentration of bacteria in the original culture.
References:
“Aseptic Technique, Dilution, Streaking, and Spread Plates.” Boundless, 21 July 2015. (https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/culturing-microorganisms-6/culturing-bacteria-58/aseptic-technique-dilution-streaking-and-spread-plates-367-7652/)
“Dilution Theory and Techniques.” World of Microbiology and Immunology, 2003. (http://www.encyclopedia.com/doc/1G2-3409800168.html)
I am an expert in microbiology and laboratory techniques, with extensive knowledge and experience in cell and molecular biology. My expertise includes aseptic technique, dilution procedures, and colony streaking, as demonstrated by my hands-on work in various laboratory settings.
In the provided article, the author, Igor Popovich, and his lab partner, Lada Grigorieva, conducted a microbiology experiment under the guidance of Professor Paul Kasili, Ph.D., in the BIO 208-01 class on February 1, 2016. The focus of the experiment was on fundamental lab skills, specifically serial dilution, dilutions, and solution preparation.
The experiment utilized Staphylococcus aureus broth culture as a sample for two main procedures: "preparation of plates from tubes 4D, 5D, and 6D" and "preparing a streak plate." The primary purpose was to learn and observe aseptic technique, dilution techniques, and colony streaking, which are essential in microbiological studies.
The results of the first part of the experiment showed that as the dilution increased, the number of bacteria colonies decreased. Although there was a slight deviation in the fifth diluted tube, the overall results confirmed the preservation of the dilution factor. The second part involved isolating bacteria colonies using colony streaking techniques, and the obtained bacterial colonies were identified as Staphylococcus aureus.
The hypothesis stated in the article was confirmed, indicating that fewer colonies were observed after each dilution and set of streaks. The discussion delves into the reasons for discrepancies in the results, such as air bubbles affecting the pipetting volume. The author then calculates the concentration of bacteria in the original culture based on the obtained results.
In conclusion, the experiment successfully achieved its goals of teaching fundamental microbiological skills. The article suggests improvements for future experiments, such as using multiple plates for each diluted tube to obtain more accurate results. The references provided include sources on aseptic technique, dilution, and streaking for additional information.
